Fluorescence assay for kinase activity

ABSTRACT

The present invention provides a sensor and methods for determining kinase activity.

RELATED APPLICATIONS

The present application is a divisional of U.S. patent application Ser.No. 10/819,587, filed Apr. 6, 2004, which is a continuation-in-part ofU.S. patent application Ser. No. 10/681,427, filed Oct. 8, 2003, nowU.S. Pat. No. 6,906,194.

FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

The subject matter of this application was developed in part withfunding from the National Institute of Health (grant application no.GM64346). The government may have certain rights in this technology.

BACKGROUND

The present invention provides sensors to monitor protein kinaseactivity continuously with a fluorescence readout. The sensor requiresminimal perturbation of a protein kinase peptide substrate. Thefluorescence response with respect to time over the course of thereaction corresponds to enzyme activity. The sensor of the presentinvention can be used in high-throughput screening of inhibitors orsubstrates, detection of activity in cell extracts or enzymepurifications, spatial or temporal localization of kinase activity in acell, and elucidation of complicated signal transduction pathways.

Protein kinases are involved in all aspects of regulation within cells.A protein kinase catalyzes the transfer of a phosphate group fromadenosine triphosphate (ATP) to a serine, threonine or tyrosine residuein a peptide or protein sequence. Each kinase is specific for the aminoacids surrounding the residue to be phosphorylated. The traditionalmethod for assaying kinase activity is discontinuous and requires32P-labelled ATP, which requires special handling. Many companies marketspecialized fluorescence kinase assay systems, all of which arediscontinuous, requiring sampling of the reaction mixture followed byadditional handling steps to label the product of the reaction with afluorescent moiety (e.g., Promega, Panvera, Calbiochem, Cell SignalingTechnology, Molecular Devices, DiscoveRx, Upstate, PerkinElmer). Acontinuous fluorescence assay that can be performed in real time is ofgreat utility. Currently, few examples of sensors capable of such assaysexist.

Approaches include: environment-sensitive fluorophores near thephosphorylation site (Wright, D. E. et al. Proc. Natl. Acad. Sci. USA1981, 78, 6048-6050; McIlroy, B. K. et al. Anal. Biochem. 1991, 195,148-152; Higashi, H. et al. FEBS Lett. 1997, 414, 55-60; Post, P. I. etal. J. Biol. Chem. 1994, 269, 12880-12887), FRET pairs flanking asequence which undergoes a conformational change upon phosphorylation(Nagai, Y. et al. Nat. Biotech. 2000, 18, 313-316; Ohuchi, Y. et al.Analyst 2000, 125, 1905-1907; Zhang, J. et al. Proc. Natl. Acad. Sci.USA 2001, 98, 14997-15002; Ting, A. Y. et al. Proc. Natl. Acad. Sci. USA2001, 98, 15003-15008; Hofmann, R. M. et al. Bioorg. Med. Chem. Lett.2001, 11, 3091-3094; Kurokawa, K. at el. J. Biol. Chem. 2001, 276,31305-31310; Sato, M. et al. Nat. Biotech. 2002, 20, 287-294; Violin, J.D. et al. J. Cell Biol. 2003, 161, 899-909), or Ca²⁺ chelation betweenthe phosphate and in internal chelator causing disruption ofPET-quenching (Chen, C.-A.; et al. J. Am. Chem. Soc. 2002, 124,3840-3841). A majority of these sensors have very modest fluorescenceincreases or sometimes decreases, with the notable exception of1.5-2.5-fold increases in the probes reported by Lawrence and coworkers(Chen 2002, supra; Yeh, R.-H.; et al. J. Biol. Chem. 2002, 277,11527-11532). However, these types of probes, with fluorophores adjacentto the phosphorylated residue or very large fluorophores may interferewith their recognition by and reactivity with certain kinases.

BRIEF SUMMARY

The present invention provides novel metal binding amino acid residuesof the formula (I) which exhibit chelation-enhanced fluorescence (CHEF)upon binding to Mg²⁺. One especially preferred amino acid residue (III)referred to as “Sox” is disclosed.

The present invention also provides peptides which include the metalbinding amino acid residues (I) of the present invention.

The present invention also provides peptidyl sensors which include themetal binding amino acid residues (I). These sensors also contain akinase recognition sequence with a hydroxyl amino acid that can bephosphorylated in the presence of a kinase. The metal binding amino acidresidue (I) is located on either side (N-terminally or C-terminally) ofthe hydro-oxyl amino acid and is preferably separated from thatrecognition sequence by a peptide which is capable of assuming a β-turnconformation (“a β-turn sequence”). In some cases, the β-turn sequenceis separated from the hydroxyl amino acid by another amino acid.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts two designs of sensors according to the presentinvention. In FIG. 1A, the metal binding amino acid is N-terminal to thephosphorylation site. In FIG. 1B, the metal binding amino acid isC-terminal to the phosphorylation site.

FIG. 2 are graphs depicting the fluorescence spectra of 10 μM each ofphosphorylated (solid line) and unphosphorylated (dashed line) peptidesin the appropriate assay mixture without enzyme: (a)Ac-Sox-Pro-Gly-(p)Ser-Phe-Arg-Arg-Arg-NH₂ (SID No: 1); (b)Ac-Leu-Arg-Arg-Ala-(p)Ser-Leu-Pro-Sox-NH₂ (SID No:2); (c)Ac-Sox-Pro-Gly-(p)Thr-Phe-Arg-Arg-Arg-NH₂ (SID No:3); and (d)Ac-Sox-Pro-Gly-Ile-(p)Tyr-Ala-Ala-Pro-Phe-Ala-Lys-Lys-Lys-NH₂ (SIDNo:4).

FIG. 3A is an high performance liquid chromatography (HPLC) trace of thereaction of Ac-Leu-Arg-Arg-Ala-Ser-Leu-Pro-Sox-NH₂ (SID No:2) (7.8 μM)by PKA after 18 min. at 30° C. FIG. 3B is an HPLC trace of the reactionof Ac-Sox-Pro-Gly-Ser-Phe-Arg-Arg-Arg-NH₂ (SID No:1) (30 μM) by PKAafter 12 min. at 30° C.

FIG. 4 are Hanes plots of Ac-Sox-Pro-Gly-Ser-Phe-Arg-Arg-Arg-NH₂ (SIDNo:1) reaction with PKC.

FIG. 5 are Hanes plots of Ac-Leu-Arg-Arg-Ala-Ser-Leu-Pro-Sox-NH₂ (SIDNo: 2) reaction with PKA.

FIG. 6 are Hanes plots of Ac-Sox-Pro-Gly-Thr-Phe-Arg-Arg-Arg-NH₂ (SIDNo:3) with PKC.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a sensor for the detecting of kinaseactivity within peptides containing kinase recognition motifs.

Sensors in accordance with the present invention are illustrated inFIG. 1. The sensor comprises a metal binding amino acid, aphosphorylation site within a kinase recognition motif, a β-turnsequence, and Mg²⁺ present in the medium.

The metal binding amino acid is of the formula (I)

where at least one R group is —SO₂X, where X is —OR″ or —NR″R′″;

R′ is hydroxy, amino, or thiol;

R″ is C₁₋₆ alkyl;

R′″ is hydrogen or alkyl; and

n is 1, 2 or 3.

The metal binding amino acids of the present invention undergochelation-enhanced fluorescence (CHEF) upon binding to Mg²⁺. Theflorescence of amino acid residues in accordance with the presentinvention increase by at least about 100%, preferably by at least about200%, more preferably by at least about 300%, when bound to Mg²⁺.

In a preferred embodiment, the metal binding amino acid is of theformula (II):

where X is —OR″ or —NR″R′″; R″ is C₁₋₆ alkyl; and R′″ is hydrogen orC₁₋₆ alkyl. Preferably X is located in the 5-position on the ringsystem. Preferably X is —NR″R′″. Preferably both R″ and R′″ are bothC₁₋₆ alkyl.

In another preferred embodiment, the metal binding amino acid is of theformula (III):

Residue (III) is referred to herein as Sox. The kinetic and fluorescenceproperties of various protein kinase substrates containing Sox invarious kinase recognition motifs is shown below in Table I. TABLE IV_(max) Target (μmol/ Fluorescence Kinase Substrate Sequence^(a) KM(μM)^(b) min/mg)^(b) Increase^(c) PKC Ac-Sox-PGS*FRRR-NH₂ 8.6 ± 29^(d) 59 ± 19^(d) 470%^(e) (SID No:1) PKA Ac-LRRAS*LP-Sox-NH₂ 1.8 ± 0.5^(f)3.7 ± 1.6^(f) 300%^(e) (SID No:2) PKC Ac-Sox-PGT*FRRR-NH₂  23 ± 3^(d)2.3 ± 0.6^(d) 280%^(g) (SID No:3) Ab1 Ac-Sox-PGIY*AAPFAKKK-NH₂ — —400%^(g) (SID No:4)^(a)Residue that is phosphorylated is marked with an asterisk (*).^(b)K_(m) and V_(max) values were obtained from initial slopes ofreaction assays, corrected appropriately for substrate and productfluorescence. Reported values are an average of four values fromseparate Hanes plots.^(c)Excitation wavelength: 360 nm, Emission wavelength, 485 nm.^(d)Assay conditions: 20 mM HEPES, pH 7.4, 10 mM MgCl₂, 0.3 mM CaCl₂, 1mM ATP, 1 mM DTT, 0.5 μg/ml phosphatidylserine, 0.1 μg/mldiacylglycerol, 0.7 nM PKC_(a)30° C.^(e)Calculated from slope (units/μM) of product and substrateconcentration versus fluorescence intensity.^(f)Assay conditions: 20 mM HEPES, pH 7.4, 10 mM MgCl₂, 1 mM ATP, 1 mMDTT, 40 units PKA, 30° C.^(g)Calculated from single peptide solutions (10 μM).

The capping group is of the formula (IV):

where at least one R group is —SO₂X, where X is —OR″ or —NR″R′″; R′ ishydroxy, amino, or thiol; R″ is C₁₋₆ alkyl; R′″ is hydrogen or C₁₋₆alkyl; and n=0, 1, 2, or 3. The capping group (IV) bonds to theN-terminus of a peptide and thus serves as an amino protecting group.

In one embodiment the N-terminus of a peptide is protected with thecapping group of the formula (IV).

Phosphorylation sites in accordance with the present invention includehydroxyl-containing amino acids within kinase recognition motifs.Examples include naturally occurring hydroxyl-containing amino acidresidues, such as serine, threonine and tyrosine, and non-naturallyoccurring hydroxyl-containing amino acid residues.

Any kinase recognition motif known in the art can be used in accordancewith the present invention. Recognition sequences with acidic residuesmay show a lessor magnitude of fluorescence increase uponphosphorylation than comparable sequences, as the affinity of theunphosphorylated peptide for Mg²⁺ increases under acidic conditions.Examples of recognition motifs which can be monitored forphosphorylation using the metal binding amino acids of the presentinvention are shown in Table II: TABLE II Kinase recognition motifProtein kinase C -Ser/Thr-Phe-Arg-Arg-Arg- (PKC) (SID No:5) cyclic-AMPdependent -Leu-Arg-Arg-Ala-Ser/Thr-Leu- kinase (PKA) (SID No:6) Abelsonkinase (Abl) -Ile-Tyr-Ala-Ala-Pro-Phe (SID No:7)

A list of other peptides which can be phosphorylated (and thecorresponding kinases) is found in Table I of Pinna & Donella-Deana,Biochemica et Biophysica Acta 1222: 415-431 (1994); incorporated hereinby reference in its entirety. Another list can be found at online atwww.neb.com/neb/tech/tech_resource/protein_tools/substraye_recognition.html(a copy of this website as it existed on Sep. 26, 2003 is provided in aninformation disclosure statement submitted concurrently with thisapplication; and is incorporated by reference in its entirety).

The hydroxyl amino acid is separated from the metal binding amino acidresidue (I) of the present invention by a dipeptidyl β-turn sequence. Inaddition to this β-turn sequence, another amino acid may also separatethe hydroxyl amino acid from the metal binding amino acid residue (1).For probes of tyrosine kinase activity, this additional amino acid istypically included in the sensor adjacent to the β-turn sequence toaccommodate the larger tyrosine side chain.

Any β-turn sequence known in the art can also be used in accordance withthe present invention. Both L-amino acids and D-amino acids can be partof the β-turn sequence. Preferably, when the metal binding amino acid islocated C-terminally to the phosphorylation site, the β-turn sequence isPro-Gly. Preferably, when the metal binding amino acid is locatedN-terminally to the phosphorylation site, the β-turn sequence isGly-Pro. Gly may be replaced with certain other amino acids to includean additional binding determinant in the sequence.

In one embodiment, the sensor comprises a sequence (SID No. 8):

-   -   H₂N—X¹—X²—X³—X⁴—COOH        where X¹ is an amino acid of the formula (I); X² and X³ are        each, independently, amino acid residues which together form a        β-turn sequence; and X⁴ is a bond or an amino acid residue. In        this embodiment, X¹ is preferably Sox, X² is Pro, and X³ is Gly.

In another embodiment, the sensor comprises a sequence (SID No. 9):

-   -   H₂N—X¹—X²—X³—X⁴—X⁵—COOH        where X¹ is an amino acid of the formula (I); X² and X³ are        each, independently, amino acid residues which together form a        β-turn sequence; X⁴ is a bond or an amino acid residue; X⁵ is a        hydroxyl containing amino acid. In this embodiment, X⁵ is        preferably the hydroxyl residue in a kinase recognition motif,        and the remainder of the kinase recognition motif is attached to        X⁵.

In another embodiment, the sensor comprises the sequence (SID No. 10):

-   -   H₂N—X³—X²—X¹—COOH        where X¹ is an amino acid of the formula (I); and X² and X³ are        each, independently, amino acids which together form a β-turn        sequence. In this embodiment, X¹ is preferably Sox, X² is Leu,        and X³ is Pro.

In another embodiment, the sensor comprises a sequence (SID No. 11):

-   -   H₂N—X⁵—X⁴—X³—X²—X¹—COOH        where X¹ is an amino acid of the formula (I); X² and X³ are        each, independently, amino acids which together form a β-turn        sequence; X⁴ is a bond or an amino acid residue; and X⁵ is a        hydroxyl containing amino acid. In this embodiment, X⁵ is        preferably the hydroxyl residue in a kinase recognition motif,        and the remainder of the kinase recognition motif is attached to        X⁵.

The sensor of the present invention can be used in a method fordetecting kinase activity. The method of the present invention comprisesproviding a sensor comprising a kinase recognition motif containing aphosphorylation site, and a metal binding amino acid of the formula (I)near to a β-turn sequence; contacting the sensor with a samplecomprising Mg²⁺, a phosphate source, and a kinase; and analyzing for thepresence of a phosphorylated peptide product.

The method of the present invention can be used in vitro or in vivo. Forin vitro applications, the reaction is typically conducted in a buffercontaining Mg²⁺ and a phosphate source. Suitable buffers include HEPESand TRIS. A preferred Mg²⁺ source is MgCl₂. A preferred phosphate sourceis ATP.

Serine/threonine and tyrosine kinases can be used in the presentinvention. Exemplary kinases include cAMP dependent protein kinase,protein kinase C, Ca/calmodulin dependent kinases, AMP activated kinase,s6 kinases, eIF-2 kinases, p34^(cdc2) protein kinase, mitogen activatedprotein kinases, casein kinase-2, casein kinase-1, glycogen sythasekinase-3, Tyr-specific protein kinases.

For in vitro applications, the concentration of kinase can range fromabout 0.5 nM to about 1 μM, typically not more than about 500 nM, andpreferably not more than about 250 nM. The concentrations of sensor canvary, but is usually ranges between about 0.1 μM to 10 mM. Adenosine5′-triphosphate (ATP) is the preferred source of phosphate, in stocksolutions of about 10-100 mM. Because most kinases have K_(m) values forATP in the range of about 10-150 μM, saturating concentrations of ATPare used to arrive at values of K_(m) and V_(max) for the substrates.

For in vivo applications, when the sensor is internalized into a cell,sufficient kinases, Mg²⁺ and phosphate sources exist in the cytosol. Forin vivo sensing, a cellular internalization sequence can be included inthe sensor design. Suitable cellular internalization sequences includePenetratins, HIV-Tat domains and poly-arginine sequences (Lindgren, M.et al. Trends Pharamol. Sci. 2000, 21, 99-103; Wadia, J. S. et al. CurrOpin. Biotech. 2002, 13, 52-56).

For applications in which the kinase is dependent on cofactors, a sourceof cofactor is also included in the sample. For example, for PKC,sources of Ca²⁺, phospholipid and diacylglycerol are needed.

The sensor of the present invention can be used to measure a kinasereaction continuously, as the metal binding amino acid residue (I) doesnot experience photobleaching.

It is therefore intended that the foregoing detailed description beregarded as illustrative rather than limiting, and that it be understoodthat it is the following claims, including all equivalents, that areintended to define the spirit and scope of this invention.

EXAMPLES Peptide Synthesis

Peptides were synthesized using standard Fmoc amino acid protectionchemistry on Fmoc-PAL-PEG-PS resin (0.22 mmol equiv.). Couplings ofFmoc-protected amino acids to the resin were carried out with1-benzotriazolyoxytris(pyrrolidino) phosphonium hexafluorophosphate(PyBOP), 1-hydroxybenzotriazole (HOBt) and diisopropylethylamine (DIEA)or O-(7-azabenzotrazol-1-yl)-1,1,3,3-tetramethyl uraniumhexafluorophosphate (HATU) and DIEA to generate the activated ester. Theresin was swelled in dichloromethane (5 min.) then DMF (5 min.) prior tosynthesis. All amino acids other than the Sox, phosphoserine,phosphothreonine and phosphotyrosine were added by the followingrepresentative procedure: removal of the Fmoc group (20% piperidinesolution in DMF, 3×5 min.), wash (DMF, 5×1 min.), coupling (aminoacid/PyBOP/DIEA, 6:6:6, 0.05 M in DMF, 45 min.), rinse (DMF, 2×1 min;DCM, 2×1 min.). To couple the Sox residue, double coupling with 2equivalents each time was used (Fmoc-Sox-OH/PyBOP/DIEA, 2:2:2, 0.15M inDMF, 2×120 min.). To couple the phosphoamino acid residues, HATU wasused (Fmoc-Xaa(PO(OBzl)OH)—OH/HATU/DIEA, 3:3:3, 0.05 M in DMF, 30 min.).

After addition of the final residue, the peptide was acetyl-capped(pyridine/acetic anhydride, 20:20, 0.15 M in DMF, 30 min.), and a finaldeblock cycle (20% piperidine in DMF, 3×5 min.) was performed to cleaveany Sox aryl ester formed. The resin was dried under high vacuumovernight prior to a 2.5-hour cleavage with trifluoroaceticacid/triisopropylsilane/water (95:2.5:2.5, 40 ml/mg resin forunphosphorylated peptides and 140 ml/mg resin for phosphorylatedpeptides).

The resulting solution was concentrated under a stream of nitrogen, andthe peptide precipitated by addition of cold 1:1 diethyl ether:hexanessolution. The pellet was triturated with cold 1:1 ether:hexanes (3×1.5ml for 15 mg resin), redissolved in water, filtered and lyophilizedovernight. Peptides were purified by preparatory reverse-phaseHPLC(C₁₈), and only fractions containing a single peak by analyticalHPLC(C₁₈) with the correct mass (ES-MS) were used for analyticalexperiments. TABLE III Characterization Data for Peptides HPLC Ret. Time[M + H]⁺ [M + H]⁺ Kinase Sequence (min) Calcd found^(c) PKCAc-Sox-Pro-Gly-Ser-Phe-Arg-Arg-Arg-NH₂ 26.7^(a) 413.2 (+3) 413.2 (SIDNo:1) Ac-Sox-Pro-Gly-pSer-Phe-Arg-Arg-Arg-NH₂ 25.7^(a) 659.3 (+2) 659.2(SID No:1) Ac-Sox-Pro-Gly-Thr-Phe-Arg-Arg-Arg-NH₂ 22.8^(b) 417.9 (+3)417.9 (SID No:3) Ac-Sox-Pro-Gly-pThr-Phe-Arg-Arg-Arg-NH₂ 21.6^(b) 666.3(+2) 666.3 (SID No:3) PKA Ac-Leu-Arg-Arg-Ala-Ser-Leu-Pro-Sox-NH₂25.9^(b) 587.8 (+2) 576.8 (SID No:2)Ac-Leu-Arg-Arg-Ala-pSer-Leu-Pro-Sox-NH₂ 25.1^(b) 627.9 (+2) 627.8 (SIDNo:2) Abl Ac-Sox-Pro-Gly-Ile-Tyr-Ala-Ala-Pro-Phe- 21.1^(c) 551.6 (+3)552.0 Ala-Lys-Lys-Lys-NH₂ (SID No:4)Ac-Sox-Pro-Gly-Ile-pTyr-Ala-Ala-Pro-Phe- 20.1^(c) 578.3 (+3) 578.3Ala-Lys-Lys-Lys-NH₂ (SID No:4)^(a)C₁₈; solvent A = water, 0.1% v/v TFA; solvent B = MeCN, 0.1% v/vTFA, 5 min. 7% B followed by linear gradient 10-50% B over 30 min.^(b)C₁₈; solvent A = water, 0.1% v/v TFA; solvent B = MeCN, 0.1% v/vTFA, 5 min. 10% B followed by linear gradient 15-50% B over 30 min.^(c)C₁₈; solvent A = water, 0.1% v/v TFA; solvent B = MeCN, 0.1% v/vTFA, 5 min. 10% B followed by linear gradient 20-70% B over 30 min.^(d)ES-MS data was collected on a PE Biosystems Mariner massspectrometer.Stock Solutions:

Due to the affinity of the phosphorylated peptides for Zn²⁺, thereagents with the highest purity and lowest metal content were used toavoid the necessity of removing metal ion impurities after preparations.

The following solutions were prepared prior to the day of the assay andstored at room temperature unless otherwise indicated:

1) Stock solution of the peptides were prepared in ultrapure (18 MΩ)water and their concentrations were determined by UV/VIS (based on thedetermined extinction coefficient of the fluorophore unit,5-(N,N-dimethylsulfonamido)-8-hydroxy-2-methylquinoline, ε=8247 M−1 cm−1at 355 nm in 0.1 M NaOH with 1 mM Na₂EDTA). An average of the valuesfrom four separate solutions, each prepared using a different volume ofthe stock solution, was read on a Beckman DU 7500 Spectrophotometer.Peptide stock solutions were stored at 4° C.

2) Magnesium chloride stock solution of about 3 M and calcium chloridestock solution of about 0.3 M were prepared from Alfa Aesar's Puratronicgrade salts. Most commercially available salts contain Zn²⁺ assignificant impurities (Thompson, R. B. et al. J. Neurosci. Methods2000, 96, 35-45) and should not be used due to the high affinity of thephosphorylated peptides for Zn²⁺. The Mg²⁺ and Ca²⁺ concentrations weredetermined by titration with a standardized solution of EDTA (Aldrich)in the presence of an Eriochrome Black T (Aldrich) (Basset, J. et al.Vogel's Textbook of Quantitative Inorganic Analysis; William Clowers:London, 1978).

3) 20 mM HEPES pH 7.4 was prepared from HEPES (SigmaUltra) adjusted topH 7.4 with sodium hydroxide (99.998+%, Aldrich) solution (1 M).

4) 20 mM HEPES pH 7.4 containing 12.5 mM MgCl₂ and 0.38 mM CaCl₂ wasprepared by addition of small volumes of stock solutions of MgCl₂ andCaCl₂ to solution 3.

5) 20 mM HEPES pH 7.4 containing 12.5 mM MgCl₂ was prepared in ananalogous way to solution 4.

6) 20 mM HEPES pH 7.4 with 5 mM dithiothreitol was prepared by firstdegassing solution 3 and then adding it to dithiothreitol (Biotechnologygrade, Mallinckrodt). The solution was stored at −80° C.

7) 100 mM ATP was prepared with adenosine 5′-triphosphate (Disodiumsalt, Low Metals Grade, Calbiochem) dissolved in ultrapure (18 MΩ) waterand the solution was stored at −80° C.

8) 10 μg/ml phosphatidylserine and 2 μg/ml diacylglycerol in 20 mM HEPESpH 7.4 was prepared by combination of appropriate volumes of chloroformsolutions of 10 mg/ml porcine brain phosphatidylserine (Avanti PolarLipids, Inc.) and 2 mg/ml 1,2-dioleoyl-sn-glycerol (Avanti Polar Lipids,Inc.). The chloroform was evaporated and solution 3 was added. Thesolution was alternated between vortexing for 3 min. intervals andincubating in a warm water bath for 1 min. for a total time of 12 min.The solution was stored at −20° C.

Assay Recipes:

PKC: On the day of the assay, a 1 μl aliquot of Protein Kinase C_(α),(Human, Recombinant, Calbiochem) was diluted with 20 μl of solution 4and stored on ice. A typical reaction contained solution 4 (84 μl),solution 6 (19 μl), solution 8 (5 μl), solution 7 (1 μl), and enzymeworking stock (1 μl). An appropriate volume of substrate stock solutionwas added to begin the reaction.

PK-A: On the day of the assay, a 1 μl aliquot of cAMP-dependent ProteinKinase (Catalytic Subunit, Mouse, Recombinant, Calbiochem) was dilutedwith 80 μl of 50 mM TRIS pH 7.5 containing 10 mM MgCl₂ and 0.3 mg/ml BSAand maintained on ice. A typical reaction contained solution 5 (90 μl),solution 6 (20 μl), solution 7 (1 μl) and an appropriate volume ofsubstrate stock solution. Enzyme working stock (1 μl) was added to beginthe reaction.

Fluorescence Experiments:

Fluorescence experiments were performed on a Fluoromax 3 from JobinYvon. 5 nm emission and excitation slit widths were used. For allexperiments, an excitation wavelength of 360 nm was used. Enzyme assayswere performed by monitoring emission at 485 nm.

Spectral Comparison of Phosphorylated and Unphosphorylated Peptides:

FIG. 2 depicts the fluorescence spectra of 10 μM each of phosphorylated(solid line) and unphosphorylated (dashed line) peptides in theappropriate assay mixture without enzyme: (a)Ac-Sox-Pro-Gly-(p)Ser-Phe-Arg-Arg-Arg-NH₂ (SID No:1) (b)Ac-Leu-Arg-Arg-Ala-(p)Ser-Leu-Pro-Sox-NH₂ (SID No:2) (c)Ac-Sox-Pro-Gly-(p)Thr-Phe-Arg-Arg-Arg-NH₂ (SID No:3) (d)Ac-Sox-Pro-Gly-Ile-(p)Tyr-Ala-Ala-Pro-Phe-Ala-Lys-Lys-Lys-NH₂ (SIDNo:4). All peptides have a maximum fluorescence emission at 485 nm, withthe exception of Ac-Leu-Arg-Arg-Ala-pSer-Leu-Pro-Sox-NH₂ (SID No:2),which has a maximum emission wavelength at 474 nm. Though this is likelyindicative of a complex forming other than a 1:1 complex, the maximumemission wavelength is constant over a wide range of peptideconcentrations with 10 mM MgCl₂.

Determination of Kinetic Constants from Fluorescence Data

To solve for K_(m) and V_(max) for this reaction, determination of theinitial rate of product formation from the increase in fluorescenceintensity is necessary. With this sensor, a correction for the decreasein fluorescence intensity due to the starting material being consumed isneeded to determine the rate of product formation from the initialslope. The fluorescence intensity at any given point can be determinedfrom the following equation:I(t)=f _(S) S(t)+f _(P) P(t)  (1)where I(t) is the fluorescence intensity, S(t) is the amount ofsubstrate in μM, P(t) is the amount of product in μM, f_(S) is thefluorescence intensity per μM) of substrate, and f_(P) is fluorescenceintensity per μM of product. The amount of substrate and product at anygiven point are related by:S(t)+P(t)=S ₀  (2)where S₀ is the initial amount of substrate. Substitution of (2) into(I) followed by rearrangement, yields: $\begin{matrix}{{P(t)} = \frac{{I(t)} - {f_{S}S_{0}}}{f_{P} - f_{S}}} & (3)\end{matrix}$

The initial velocity of the reaction is the change in the amount ofproduct over time, so taking the derivative of (3) with respect to timegives: $\begin{matrix}{v = {\frac{\mathbb{d}{P(t)}}{\mathbb{d}t} = \frac{\frac{\mathbb{d}{I(t)}}{\mathbb{d}t}}{f_{P} - f_{S}}}} & (4)\end{matrix}$

The initial slope of the reaction, dI(t)/dt, was measured within thefirst 5% of substrate turnover. The constants f_(P) and f_(S) werecalculated from the slope of a line of fluorescence intensity versusconcentration of P and S, respectively. A linear fit of a Hanes plot([S]/V vs. V) was used to find K_(m) and V_(max).

HPLC and MS Data for Kinase Reactions:

All reactions were followed in the fluorometer and quenched with 40 μlof a 0.1 M Na₂EDTA stock solution and then stored on ice. FIG. 3Adepicts the phosphorylation of Ac-Leu-Arg-Arg-Ala-Ser-Leu-Pro-Sox-NH₂(SID No:2) (7.8 μM) by PKA after 18 min. at 30° C. FIG. 3B depicts thephosphorylation of Ac-Sox-Pro-Gly-Ser-Phe-Arg-Arg-Arg-NH₂ (SID No:1) (30μM) by PKA after 12 min. at 30° C. The HPLC peak identities for thispeptide are shown below in Table 3. TABLE 3 HPLC peak identities of SIDNos. 1 and 2 HPLC Peak T_(R) Expected Peptide Kinase No. (min.) [M +xH]^(x+) [M + xH]^(x+) Identification PKA 1 24.8^(a) 627.8 (2+) 627.8Product (SID No. 2) PKA 2 26.1^(a) 587.8 (2+) 587.8 Substrate (SID No.2) PKC 1 32.5^(b) 659.2 (2+) 659.2 Product (SID No. 1) PKC 2 33.4^(b)413.2 (3+) 413.2 Substrate (SID No. 1)^(a)C18; solvent A = water, 0.1% v/v TFA; solvent B = MeCN, 0.1% v/vTFA, 10 min. 0% B, linear gradient 0-10% B over 2 min. followed bylinear gradient 10-50% B over 30 min.^(b)C18; solvent A = water, 0.1% v/v TFA; solvent B = MeCN, 0.1% v/vTFA, 10 min. 15% B followed by linear gradient 15-50% B over 30 min.

Comparison of Fluorescence and HPLC Data Concerning Amount of ProductFormed: ${P(t)} = \frac{{I(t)} - {f_{S}S_{0}}}{f_{P} - f_{S}}$ [Product][Product] Initial [Product] (μM) from (μM) from Kinase Substrate Lengthof (μM) from HPLC HPLC Reaction Conc. reaction fluorescence (228 nm)(280 nm) PKA  46 μM 300 sec. 1.6 1.3 1.9 PKA 7.8 μM 700 sec. 3.4 3.3 3.5PKA 7.8 μM  18 min. 4.6 4.4 4.8 PKC  30 μM  12 min. 3.3 3.2 4.7Hanes Plots:

FIG. 4 are Hanes plots of Ac-Sox-Pro-Gly-Ser-Phe-Arg-Arg-Arg-NH₂ (SIDNo:1) reaction with PKC. K_(m) and V_(max) values were determined usingf_(S,avg)=1.1×105±0.3 units/μM and f_(P,avg)=6.3×105±1.0 units/μM.

FIG. 5 are Hanes plots of Ac-Leu-Arg-Arg-Ala-Ser-Leu-Pro-Sox-NH₂ (SIDNo:2) reaction with PKA. K_(m) and V_(max) values were determined usingf_(S,avg)=2.5×10⁵±0.1 units/μM and f_(P,avg)=9.9×10⁵±0.2 units/μM.

FIG. 6 are Hanes plots of Ac-Sox-Pro-Gly-Thr-Phe-Arg-Arg-Arg-NH₂ (SIDNo:3) reaction with PKC. K_(m) and V_(max) values were determined forusing Fs and Fp values determined the day of each experiment.

1. A method for detecting kinase activity, comprising the steps of: (a)providing a peptide comprising: (1) a kinase recognition sequencecontaining a phosphorylation site; (2) a β-turn sequence; and (3) anamino acid of the formula (I):

where each R is independently hydrogen or —SO₂X, wherein at least one Rgroup is —SO₂X, where X is —OR″ or —NR″R′″; R′ is hydroxy, amino, orthiol; R″ is C₁₋₆ alkyl; R′″ is hydrogen or alkyl; and n is 1, 2 or 3;(b) contacting the peptide with a sample comprising Mg²⁺, a phosphatesource, and a kinase; and (c) analyzing for the presence of aphosphorylated peptide product.
 2. The method of claim 1, wherein theamino acid is of the formula (II):

where X is —OR″ or —NR″R′″; R″ is C₁₋₆ alkyl; and R′″ is hydrogen orC₁₋₆ alkyl.
 3. The method of claim 1, wherein the amino acid is of theformula (III):


4. The method of claim 1, wherein fluorescence of the peptide increasesby at least about 100% when bound to Mg²⁺.
 5. The method of claim 1,wherein the K_(m) of the peptide for the kinase is in the micromolarrange.